An upstream element containing an ETS binding site is crucial for transcription of the human presenilin-1 gene

Deletion mapping of the human presenilin-1 (PS1) promoter delineated the mo st active fragment from -118 to +178 in relation to the transcription start site mapped in this study, in both human neuroblastoma SK-N-SH and hepatom a HepG2 cells. 5' deletions revealed that a crucial element controlling ove r 90% of the promoter activity in these cell lines is located between -22 a nd -6, A mutation altering only two nucleotides of the ETS consensus sequen ce present at -12 (GGAA to TTAA) has a similar effect. Electrophoretic mobi lity shift assays showed that a set of specific complexes between nuclear f actors and the PS1 promoter are eliminated by this point mutation, as well as by competition with an ETS consensus oligonucleotide. Competition experi ments in DNase I footprinting correlated with electrophoretic mobility shif t assays and showed that only one of several footprints over the PS1 promot er is eliminated by competition with an ETS consensus oligonucleotide. It e xtends from -14 to -6 and surrounds the ETS motif present at -12, Thus, a c rucial ETS element is present at -12 and binds a protein(s) recognizing spe cifically the ETS consensus motif. At least one such complex is eliminated by preincubating the nuclear extract with an antibody with broad cross-reac tivity with Ets-l and Ets-a proteins, thus confirming that an ETS transcrip tion factor(s) recognizes the -12 motif. Several Spl binding motifs at posi tions -70, -55, and +20 surround this ETS element. Competition DNase I foot printing showed that Spl-like nuclear factors recognize specifically these sites in both cell lines. Furthermore, a combination of 5' and 3' deletions indicated the presence of positive promoter elements between -96 and -35 a s well as between +6 and +42. Thus, transfection and footprinting assays co rrelate to suggest that Spl transcription factor(s) bind at several sites u pstream and downstream from the initiation site and activate the transcript ion of the PS1 promoter. Sequences downstream from the transcription initia tion site also contain major control elements. 3' deletions from +178 to +1 07 decreased promoter activity by 80%. However, further deletion to +42 inc reased promoter activity by 3-4-fold. Collectively, these data indicate tha t sequences upstream and downstream from the transcription start site each control over 80% of the promoter activity. Hence, this suggests that protei n-protein interactions between factors recognizing downstream and upstream sequences are involved.